Knauer Clarity 8.2.2 Extensions MS Owner's manual

MS EXTENSION

Clarity Extension ENG

Code/Rev.: M168/80D

Date: 4/1/2019

Phone: +420 251 013 400 DataApex Ltd.

Fax: +420 251 013 401 Petrzilkova 2583/13

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trademarks of Microsoft Corporation.

DataApex reserves the right to make changes to manuals without prior notice. Updated manuals can be

downloaded from www.dataapex.com.

Author: DM

MS Extension Table of Contents

Contents

1 MS - Mass Spectroscopy 1

2 Specification 2

3 Installation 3

4 Key Features 4

4.1 Basic principles and terms 5

5 MS Extension Description 6

5.1 MS Chromatogram window 6

5.1.1 MS Menu 9

5.1.1.1 MS Search 13

5.1.1.2 Add MS Spectrum to Library 22

5.1.1.3 Mean and Backgrounds 23

5.1.1.4 Create Spectrum Label 25

5.1.2 Result Table 28

5.1.3 Measurement Conditions - MS Method 30

5.1.4 Integration Table 37

5.1.5 Import MS Chromatogram 39

5.1.6 Export MS Chromatogram 42

5.1.7 Graph Properties - MS Spectra tab 44

5.2 Method Setup 46

5.2.1 Method Setup - MS Method 46

5.2.2 Method Setup - Integration 47

5.3 MS Calibration 48

5.4 Data Acquisition 49

5.5 Report Setup - MS 50

6 Clarity MS operation 52

6.1 Setting MS Instrument 53

6.2 Opening MS demo project 53

6.3 Setting MS Search position 53

6.3.1 Creating and filling your own library 54

6.3.2 Managing MS Library 55

6.4 Creating MS Method 56

6.4.1 Using Single Compound Search 57

6.4.2 Using Automatic Compound Search 57

6.4.3 Using Target Compound Search 58

6.4.4 Editing MS Method 58

6.5 Defining Reference m/z values 59

6.6 Creating of the MS Calibration 60

6.7 Importing MS Chromatogram 61

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MS Extension Table of Contents

To facilitate the orientation in the MS Extension manual and Clarity chromatography station,

different fonts are used throughout the manual. Meanings of these fonts are:

Instrument (blue text) marks the name of the window to which the text refers.

Open File (italics) describes the commands and names of fields in Clarity, parameters that can

be entered into them or a window or dialog name (when you already are in the topic describing

the window).

WORK1 (capitals) indicates the name of the file and/or directory.

ACTIVE (capital italics) marks the state of the station or its part.

The bold text is sometimes also used for important parts of the text and the name of the Clarity

station. Moreover, some sections are written in format other than normal text. These sections are

formatted as follows:

Note: Notifies the reader of relevant information.

Caution: Warns the user of possibly dangerous or very important information.

▌ Marks the problem statement or trouble question.

Description: Presents more detailed information on the problem, describes its causes,

etc.

Solution: Marks the response to the question, presents a procedure how to remove it.

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MS Extension 1 MS - Mass Spectroscopy

1 MS - Mass Spectroscopy

The Clarity MS Extension is an optional part of the Clarity

Chromatography Station (from version 5.0). It is intended for data

acquisition and evaluation of data measured on MS Detectors.

MS Extension is also compatible with Clarity Offline software.

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MS Extension 2 Specification

2 Specification

The MS Extension is an optional, fully integrated part of Clarity software.

It can be ordered as a part of new datastation or as an Extension to

existing datastation (p/n A38).

Note: MSExtension has higher demands on your PCconfiguration. We

recommend PC with SSDdrive installed. Please check recommended

PCconfiguration listed in the D016 Datasheet - Compatibility Table.

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MS Extension 3 Installation

3 Installation

The MS Extension is enabled by appropriate user code entered during

installation or later by using the Help - User Code command from the

Clarity main window.

To switch an Instrument to a mode where it can collect and evaluate MS

data, select the LC-MS or GC-MS option from the Instrument Type Setting

dialog.

Instrument Type Setting dialog is invoked by clicking on the button in

the System Configuration dialog.

Options that are technically possible and have been purchased are

enabled by default. Otherwise they are automatically disabled.

Fig 1: Setting up the LC-MS Instrument

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MS Extension 4 Key Features

4 Key Features

The MS Extension brings the following features to the Clarity station:

MS Chromatogram window - standard Clarity Chromatogram window

was modified to display the spectral information too. The graph pane now

holds one more graph showing the spectrum at any given time or at a time

span, which is noted in the top-right corner in the legend area.

MS Method - MS method allows the user to select the extracted signals on

which the compounds will be identified and quantified. Each compound

may use different reference signals, so that more than one compound may

be identified in any part of the chromatogram.

Library search -Clarity MS Extension allows the user to use spectral

libraries for peak identification and/or compound confirmation.

NIST Libraries -Clarity uses the NISTLibrary format for storing spectra,

and works flawlessly with commercial NIST Libraries or those created by

the user.

Automatic Compound Search -Clarity allows to search any selected

signal automatically for the presence of spectra that are defined in the

spectral library.

The following standard Clarity features will also help the user to make the

best of the MS analysis in Clarity:

User calculations - the user can define custom calculations in the Result

Table and Summary Table. With the use of the integrated editor you can

create your own columns from the original ones and use individual

mathematical functions.

Reports - user selectable report sections, WYSIWYG formatting of Graphs

and Tables, and the possibility to report the spectral information for any

peak in the chromatogram.

Batch - automatic batch processing, display, export or print of any number

of chromatograms.

Summary Result Table - displays and prints selected results from all

simultaneously displayed chromatograms.

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MS Extension 4 Key Features

4.1 Basic principles and terms

Mass Spectrometry (MS ) is an analytical detection method that

determines the structure of compounds from the ions to which the

compound is fragmented.

m/z means Mass to Charge ratio. It is the value particular ions are divided

by. Most common usage is the usage of ions with the charge of +1 or -1,

making the m/z effectively just the mass of the ion.

MS Detector is the device serving for the Mass Spectrometry . It

generally composes of several parts - Sample Inlet,Ion Source where the

compounds entering the detector are fragmented to ions, Mass Analyzer

in which the particular ions are separated based on their m/z ratio and

Detector, where the ions are detected.

MS Spectrum is the basic information coming out of the MS Detector - it

is a set of data describing the relative intensities of ions of particular m/z

fragmented from the compound.

Raw Data is a matrix of the spectra and the retention time.

Total Ion Current (TIC) represents the total signal coming from the MS

Detector, in fact summing all the individual ion intensity values from each

spectra to single value.

Base Peak Intensity (BPI) is another interesting signal in the MS practice

- it represents the signal showing the intensity of the highest intensity ion

from each spectra.

Extracted Ion Current (EIC) is the signal over time of one single ion of

selected m/z, as gained from the Raw Data.

Raw Spectrum is a continuous spectrum (similar to the UV spectrum) of

the compound - individual data points are connected by a curve.

Stick Spectrum is a simplified version of the Raw Spectrum , where

"Peaks" made by small imprecission of the MS Detector are instead

represented by a single m/z value, so called Stick.

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MS Extension 5 MS Extension Description

5 MS Extension Description

After installation, new functions of the MS Extension will be available.

Only features changed or added to the Clarity standard mode are listed

and described here.

5.1 MS Chromatogram window

On the GC- MS and LC- MS Instrument the standard Chromatogram

window was replaced by MS Chromatogram window. The functionality of

MS Chromatogram window differs slightly from the functionality of the

Chromatogram window, but most of the functions are common between

both windows.

Fig 2: MS Chromatogram window

Graph pane is divided into two parts in the MS Chromatogram window -

original graph as in standard Chromatogram window, and the spectrum

part displaying the spectrum at any given time or time interval. There is a

special mode called MS Offsetting for displaying the signals in the MS

Chromatogram window, allowing to offset different signals to the same

baseline level while keeping the ratios of the signals, which can be set in

the Graph Properties - Signals dialog invoked by using the Properties…

command from the local menu. This setting helps to eliminate the

background noise on some signals (notably TIC) for optical evaluation of

correct ion ratios. The settings for the behavior of the spectral graph can

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MS Extension 5 MS Extension Description

be selected using the Graph Properties - MS Spectra dialog invoked by

using the Properties… command from the local menu.

Signals in MSExtension

The number of signals displayed in the graph will change contextually as

the user changes focus in the graph itself or in the Result Table. Each

peak in the MS Extension is defined by up to four EIC X - Reference m/z X

signals and a Quantification signal, which may be TIC, any of the

Reference signals, sum of those or even an external signal. Different

peaks may (and indeed will) differ in number of such defined signals and

in the signals themselves - the EIC - reference m/z for them will differ too.

Order under which the signals are displayed is defined by the signals set

in the System Configuration (and later Method Setup ) that were used

during the acquisition of the chromatogram. Standard signals defined in

the System Configuration and Method Setup dialogs will be displayed

first. Next, if a peak is selected, will be it's quantification signal, followed by

the signals of reference ions. If one of the reference ions is at the same

time used as a quantification signal, it will not be displayed for the second

time. Next in order will be any signals displayed by the user from the raw

data.

The standard signals behave exactly as the users are used to in Clarity,

and they are saved when the chromatogram is saved. The rest of the

signals are so called "Temporary Signals" created according to the

information set in the MS Method and created always fresh from the raw

data when they are needed. If an MS chromatogram is opened on non-MS

Clarity Instrument, only the standard signals will be available for

inspection and work, but the temporary signals will not.

Result Table

Another major change in the MS Chromatogram window touches the

Result Table . On non- MSInstruments each signal has it's own Result

Table, whereas on MS Instrument, the Result Table is common through

all signals. The table header which normally changes color to match the

color of the active signal is black on MS Instrument regardless of the

signal selected.

The Result Table is a combination of two separate result tables - one

created according to the MS Method Table, second one created according

to the selected standard signal. The information on the signal used for this

purpose is stored in the header of the table and can be changed by using

the Result Table Signal option on the upper part of the Measurement

Conditions - MS Method tab.

Other changes in MS Chromatogram window

All changes in the MS Chromatogram window are described on particular

places - MS menu,Result Table,Measurement Conditions - MS Method

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MS Extension 5 MS Extension Description

tab,Integration Table,Import Chromatogram and the Graph Properties -

MS Spectra tab.

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MS Extension 5 MS Extension Description

5.1.1 MS Menu

Menu containing all commands available in the MS Extension.

MS Method Switches the display of the lower part of the MS

Chromatogram window to the Measurement

Conditions - MS Method tab.

Set NIST Libraries

Directory…

Allows to set the correct location of the NIST

Libraries, if they are installed on the computer.

Clarity installation includes also basic NIST demo

libraries which are stored in the \UTILS\NIST\MS

SEARCH subdirectory of the Clarity working

directory (C:\CLARITY\BIN by default), which can be

used instead.

Note: It is necessary to select the folder where the library names are

displayed as subdirectories for Clarity to function.

Select Spectral Data Command for working with multiple spectra in a

single chromatogram. Opens a sub-menu allowing

to switch to different spectrum. The same action may

be also performed from the select-box in the MS

toolbar.

Prefer Spectral Data

from Method

Command for working with multiple spectra in a

single chromatogram. Sets whether for compounds

already present in the MS Method the spectrum

displayed will be the spectrum selected in the

method.

Single Compound

Search…

Opens the MS Search dialog on the Single

Compound Search tab. More information on the

Single Compound Search tab can be found in the

chapter "MS Search" on pg 13.

Automatic Compound

Search…

Opens the MS Search dialog on the Automatic

Compound Search tab. More information on the

Automatic Compound Search tab can be found in

the chapter "MS Search" on pg 16.

Target Compound

Search…

Opens the MS Search dialog on the Target

Compound Search tab. More information on the

Target Compound Search tab can be found in the

chapter "MS Search" on pg 19.

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MS Extension 5 MS Extension Description

Manage Libraries… Opens the MS Search external program which

allows to create user libraries, manage them and

copy compound spectra to them.

Add Spectrum to

Library…

Sends the selected spectrum to the MS Search

external program via Add MS Spectrum to Library

dialog described on pg 22.

Show Spectrum Allows to display a spectrum at any given time or

any time interval. Invoking this command locks the

cursor in the graph pane - just clicking on the time

shows the spectrum at that point, pressing Ctrl and

clicking twice allows to select the time interval

through which the spectrum will be averaged.

Add Reference m/z When you are in a MS Method Table row, using this

command locks the cursor in the spectrum graph.

Clicking on any m/z stick adds the given m/z ion as a

reference m/z for the compound. When all four

reference m/z ions are filled in, you need to delete

one of the four reference m/z ions before adding a

new one.

Add Compound w/o

Library Spectrum…

When invoked, locks the cursor in the chromatogram

graph pane. Clicking on any time will invoke the Add

Compound w/o Library Spectrum dialog described

in the section Add Compound w/o Library

Spectrum on pg 12

Add Selected m/z

Signal as Temporary

Using this function locks the cursor in the spectrum

graph. Clicking on any m/z will add the extracted

signal of that m/z to the chromatogram if maximum

signals in the chromatogram is not achieved yet.

Add Temporary m/z

Signal - Manually…

Using this function invokes the Add Temporary m/z

Signal Manually dialog described in the section Add

Temporary m/z Signal - Manually… on pg 12

allowing to extract the signal on any chosen m/z

from the raw data if maximum signals in the

chromatogram is not achieved yet. It is possible to

get a signal for the range of several defined m/z

values.

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MS Extension 5 MS Extension Description

Note: Maximum of 24 signals is allowed in any MS chromatogram. This

includes TIC, BPI, external signals, reference signals and any

extracted signals using Add Selected m/z Signal as Temporary

or Add Temporary m/z Signal - Manually… function, but not the

Quantification signal.

Remove All

Temporary m/z

Signals

Serves for removing the no-longer necessary

temporary added signals from the display in the MS

Chromatogram window. All temporary added

signals will be closed upon invoking this command.

Mean and

Backgrounds

Command opening sub-menu with commands used

for setting the mean and background evaluation

intervals.

Set Mean Calculation

Interval

Using this function locks the cursor in the MS

chromatogram graph. Clicking on any time point

sets the start of the mean calculation interval,

clicking for the second time selects the end of the

mean calculation interval. Both values are inserted

into the MS method of the chromatogram for the

given peak in the format specified in Mean and

Backgrounds dialog.

Set Background 1

Interval

Using this function locks the cursor in the MS

chromatogram graph. Clicking on any time point

sets the start of the background 1 interval, clicking

for the second time selects the end of the

background 1 interval. Both values are inserted into

the MS method of the chromatogram for the given

peak in the format specified in Mean and

Backgrounds dialog.

Set Background 2

Interval

Using this function locks the cursor in the MS

chromatogram graph. Clicking on any time point

sets the start of the background 2 interval, clicking

for the second time selects the end of the

background 2 interval. Both values are inserted into

the MS method of the chromatogram for the given

peak in the format specified in Mean and

Backgrounds dialog.

Configure Mean and

Backgrounds…

Opens the Mean and Backgrounds dialog used for

defining the defaults for mean calculation interval

and background intervals.

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MS Extension 5 MS Extension Description

Create Spectrum

Label

Command serving for creating labels in MS spectral

graph. Two options enable to create either Text

labels or Line labels (for more details see the

chapter Create Spectrum Label on pg 25).

Add Compound w/o Library Spectrum

Fig 3: Add Compound w/o Library Spectrum dialog

The dialog allows to enter a new row into the MS Method Table. Selecting

a unique Compound name is necessary.

Add Temporary m/z Signal - Manually…

Fig 4: Add Temporary m/z Signal - Manually…

The dialog invoked after using the Add Temporary m/z Signal -

Manually… command allows to extract the desired m/z signal from the raw

data. Any values complying with spectral precision are allowed to be

entered into the dialog, the values can be input as single values

separated by semicolon (;) or as a range using dash (-). E.g.: 45;62;100-

125.

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MS Extension 5 MS Extension Description

5.1.1.1 MS Search

MS Search dialog serves for setting the parameters for the search in

spectral libraries. Clarity can perform three types of search - Single

Compound Search,Automatic Compound Search and Target Compound

Search.

Single Compound Search

Single Compound Search tab serves for performing a simple search of a

single spectrum in one or more spectral libraries. It is invoked by using the

Single Compound Search command from the MS menu of the MS

Chromatogram window.

Fig 5: MS Search - Single Compound Search dialog

Search In Ret. Time

Either an exact retention time or an interval can be selected to search in.

The values can be input manually or by selecting in the graph. The

spectrum from the selected retention time will be compared with the

spectral libraries and results will be returned based on further setting, i.e.

Search Options and Background Subtraction.

Select From Graph

When this button is clicked, a mouse cursor will be focused into the graph,

where either an exact retention time can be selected by one click into the

graph, or a range of retention time can be selected: while holding the

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MS Extension 5 MS Extension Description

CTRL key, first click into the graph to select the beginning of the desired

interval, and second click into the graph to select the end of the desired

interval.

Search Options

Section allowing to set several parameters for the spectral search.

Min Match Factor

Restricts the hits returned to Clarity from MS Search program to just

those with match factor higher than or equals to the value set in the

field. Valid numbers are whole numbers from 0 to 1000.

Max Hits

Restricts the number of hits returned from the MS Search program to

Clarity. Changing the value in the field only applies when the search

is invoked again. Valid values are whole numbers from 1 to 100.

Restrict m/z Range

Sets the range of m/z values that will be searched in the library

spectra.

Use Selected m/z

lSearch Only Selected performs the search using only up to

four selected m/z values. Using this option searches the

libraries only for spectra containing the selected m/z ions with

non-zero intensities, giving best matches to compounds that

have the relative intensities of such sticks closest to ones in the

chromatogram.

Note When the Restrict m/z Range together with

Search Only Selected are set, only those m/z

ions belonging to this restricted range are

searched.

lSearch All But Selected excludes from the search up to four

ions selected in the m/z 1..4 fields.

Background Subtraction

Up to two backgrounds can be selected to be subtracted from the

spectrum before the search. Such backgrounds can be input either as an

exact retention time or as an interval.

Select From Graph

When this button is clicked, a mouse cursor will be focused into the graph,

where either an exact time for background subtraction can be selected by

one click into the graph, or a range of time for background subtraction can

be selected: while holding the CTRL key, first click into the graph to select

the beginning of the desired interval, and second click into the graph to

select the end of the desired interval.

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MS Extension 5 MS Extension Description

Search

When invoked, the search in NIST Libraries is performed using the

external MS Search program. The MS Search being searched in can be

set in the MS Search program (menu options Options - Library Search

Options - Libraries tab), along with other search parameters.

Preview Spectrum in Library

When invoked, the selected spectrum will be shown in NIST Libraries

using the external MS Search program. More parameters can be set in

the MS Search program (menu Options - Library Search Options -

Libraries tab).

Caution: Parameters changes in the MS Search - Single Compound Search dialog

will not be applied until the Search or Preview Spectrum in Library button

is pressed once again.

Add Selected to Method

Adds the checked rows from MS Library Search into the MS Method table.

MS Library Search table

This table contains the results of the search from the MS Search program.

The table contains the Match ,Reverse Match ,Probability,Compound

Name,Library,ID,Formula,MW and CAS No. columns, as used in the MS

Search program.

Spectrum graph

The graph displays the overlay of the chromatogram signal as set in the

Search In Ret. Time field used for the library search and the library

spectrum of a compound selected in the MS Library Search table.

Standard operations (zooming, etc.) are allowed in the graph, it's format

(mainly peak tag format, overlay/head- to- tail mode, spectra colors) is

dependent on the settings on the Graph Properties - MS Spectra tab. It is

possible to open the tab using the context menu invoked by right-click in

the graph area.

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MS Extension 5 MS Extension Description

Automatic Compound Search

Automatic Compound Search tab serves for performing an initial search

on a chromatogram where the user knows what he can expect. It is

possible to quickly fill the whole MS Method table by running an

Automatic Compound Search . The function is invoked by using the

Automatic Compound Search command from the MS menu of the MS

Chromatogram window.

Fig 6: MS Search - Automatic Compound Search dialog

Use Signal

Sets the signal on which the peaks will be detected and their spectra will

be matched with spectral libraries. It is possible to select any standard

signal including TIC , as well as external signals and signals extracted

from the raw data (in such case you have to select the desired m/z in the

appropriate field). While adding found compounds to MS Method, the

signal set in this combo box will be propagated to Quantify On column in

MS Method table.

Search In Ret. Time

By default the search interval is in the whole chromatogram. If needed,

such search interval can be specified in the from and to edit boxes,

enabled after unchecking the check box Whole Chromatogram . The

spectrum from the selected retention time will be compared with the

spectral libraries and results will be returned based on further setting, i.e.

Search Options.

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Knauer Clarity 8.2.2 Extensions MS Owner's manual

Knauer Clarity 8.2.2 Extensions MS Owner's manual

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